1. Field of the Invention
The present invention relates to the addition of histidine amino acids to the cytoplasmic domains of membrane and viral envelope proteins for the purposes of solubilizing, and/or reconstituting viral envelope proteins in lipid containing vesicles. The invention further relates to the use of GP64-6His to catalyze delivery of therapeutic, genetic, or antigenic compounds via fusion of lipid membranes or bilayers
2. Background Art
Enveloped viruses infect cells by one of two membrane fusogenic processes. The first is direct cell surface membrane fusion or pH-independent membrane fusion. Briefly, contact with the appropriate cell surface receptor activates viral envelope proteins to catalyze membrane fusion of the virus with the cytoplasmic membrane resulting in deposition of the infectious nucleocapsid inside the cell. A second class of enveloped viruses enters by receptor-mediated endocytosis or pH-dependent processes. pH dependent viruses also target their host cell by interaction with a specific receptor. However rather than fusing directly at the cell surface, these viruses remain attached to the receptor which is subsequently internalized within an endosomal vacuole derived from the cell surface lipids. This vacuole, termed an endosome, enters the lysosome. The lysosome is an acidic (low pH) cellular compartment which metabolizes, recycles, and breaks down endosomal contents. When pH-dependent enveloped viral proteins are exposed to the low pH of the lysosome, a conformational change occurs resulting in the activation of membrane fusion. These proteins then mediate fusion of the viral envelope with the lipid membrane to escape the lysosome. As a result, the virus is then free to replicate within the host cell.
Liposomes are small vesicles composed of phospholipid bilayers of varying compositions. As such they can be used to encapsulate, protect, and deliver therapeutic compounds, DNA, RNA (sense or antisense), or proteins to cells. Currently liposomal encapsulation systems have been of limited utility. There are two primary reasons for this: 1) While liposomes do in fact enter cells, they do so primarily by endocytosis. As discussed previously this process results in deposition of endosomal vacuoles within the lysosome. The acidic nature of the lysosome results in degradation or destruction of the liposomal contents, in most cases, 2) Liposomes, unlike viruses, lack the ability to discriminate between different cell and tissue targets, since they do not bind to specific receptors on the cell surface.
GP64 is a class I membrane glycoprotein, which constitutes the major envelope protein of the AcMNPV budded baculovirus virion (Volkman and Goldsmith, 1984; Volkman et al., 1984). This protein is both necessary and sufficient for mediating pH-dependent membrane fusion (Blissard and Wenz, 1992), and is essential for cell-cell transmission of the enveloped virion (Monsma et al., 1996).
When GP64 (or GP64-6His) is expressed on the cell surface of Sf9 insect cells, host and target cell membranes can be fused (syncytia formation) simply by application of acidic buffer solution (phosphate buffer saline or citrate buffer pH 5.5-4.8) This invention provides functionally competent GP64-6His protein to create acid-activated fusion competent liposomes for various biomedical and research purposes.